Molecular Basis of Phosphorylation-Induced Activation of the NADPH Oxidase

Groemping, Yvonne; Lapouge, Karine; Smerdon, Stephen J.; Rittinger, Katrin

doi:10.1016/s0092-8674(03)00314-3

Cited by 357 publications

(442 citation statements)

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“…In the resting state, the ternary complex of p47 phox , p67 phox , and p40 phox is in the cytoplasm. p47 phox is folded and inactive, due to inhibitory intramolecular interactions Groemping et al, 2003;Durand et al, 2006). p40 phox is also folded and inactive due to inhibitory intramolecular head-to-tail (PX-PB1) interactions.…”

Section: Discussionmentioning

confidence: 99%

“…masking both the N-and C-terminal SH3 domains and the PX domain Karathanassis et al, 2002;Groemping et al, 2003;Durand et al, 2006). When phagocytes are activated after cell stimulation, the autoinhibitory region (AIR) (aa 286-340) is phosphorylated, thereby disrupting the intramolecular interactions and rendering both SH3 domains and the PX domain accessible to bind p22 phox and membrane lipids, respectively (Karathanassis et al, 2002;Ago et al, 2003;Groemping et al, 2003) . It was reported that the interaction between the PR motif in the PX domain and the C-terminal SH3 domain of p47 phox .…”

Section: Pb1 Domain Of P40 Phox Masks the Px Domain Of P40 Phoxmentioning

confidence: 99%

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A Regulated Adaptor Function of p40^phox: Distinct p67^phoxMembrane Targeting by p40^phoxand by p47^phox

Ueyama

Tatsuno

Kawasaki

et al. 2007

MBoC

101

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In the phagocytic cell, NADPH oxidase (Nox2) system, cytoplasmic regulators (p47 phox , p67 phox , p40 phox , and Rac) translocate and associate with the membrane-spanning flavocytochrome b 558 , leading to activation of superoxide production. We examined membrane targeting of phox proteins and explored conformational changes in p40 phox that regulate its translocation to membranes upon stimulation. GFP-p40 phox translocates to early endosomes, whereas GFP-p47 phox translocates to the plasma membrane in response to arachidonic acid. In contrast, GFP-p67 phox does not translocate to membranes when expressed alone, but it is dependent on p40 phox and p47 phox for its translocation to early endosomes or the plasma membrane, respectively. Translocation of GFP-p40 phox or GFP-p47 phox to their respective membrane-targeting sites is abolished by mutations in their phox (PX) domains that disrupt their interactions with their cognate phospholipid ligands. Furthermore, GFP-p67 phox translocation to either membrane is abolished by mutations that disrupt its interaction with p40 phox or p47 phox . Finally, we detected a head-to-tail (PX-Phox and Bem1 [PB1] domain) intramolecular interaction within p40 phox in its resting state by deletion mutagenesis, cell localization, and binding experiments, suggesting that its PX domain is inaccessible to interact with phosphatidylinositol 3-phosphate without cell stimulation. Thus, both p40 phox and p47 phox function as diverse p67 phox "carrier proteins" regulated by the unmasking of membrane-targeting domains in distinct mechanisms. INTRODUCTIONIn phagocytic cells, reactive oxygen species (ROS) are produced by NADPH oxidase (Nox2 system). The enzyme is a multiprotein complex assembled from a membrane-spanning flavocytochrome b 558 (composed of gp91 phox [Nox2] and p22 phox ) and four cytoplasmic components (p47 phox , p67 phox , p40 phox , and Rac) (Leto, 1999;Nauseef, 2004;Quinn and Gauss, 2004). In unstimulated phagocytes, the oxidase is dissociated and inactive: the flavocytochrome b 558 is stored on the membranes of intracellular granules (Jesaitis et al., 1990), Rac is maintained in a GDP-bound cytoplasmic complex dimerized with Rho-guanine nucleotide dissociation inhibitor (GDI) , and the other phox proteins associate in a separate ternary cytoplasmic complex (p47 phox -p67 phox -p40 phox ) in a dephosphorylated state (Bolscher et al., 1989;Rotrosen and Leto, 1990;Kuribayashi et al., 2002;Lapouge et al., 2002). During phagocyte activation, intracellular granules containing flavocytochrome b 558 fuse with phagosomes; p47 phox is phosphorylated, thereby inducing conformational changes in p47 phox that promote the interaction of the cytoplasmic complex with the flavocytochrome b 558 ; and Rac dissociates from Rho-GDI and translocates independently to the membrane after exchange of GDP for GTP (Heyworth et al., 1994;Zhao et al., 2003), resulting in generation of superoxide anion by the transfer of electrons from cytoplasmic NADPH to molecular oxygen.Chronic granulomatous disease...

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Section: Discussionmentioning

confidence: 99%

Section: Pb1 Domain Of P40 Phox Masks the Px Domain Of P40 Phoxmentioning

confidence: 99%

A Regulated Adaptor Function of p40^phox: Distinct p67^phoxMembrane Targeting by p40^phoxand by p47^phox

Ueyama

Tatsuno

Kawasaki

et al. 2007

MBoC

101

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“…One required step for NADPH oxidase activation is the phosphorylation of p47 phox by PKC, which permits p47 phox to interact with the cytoplasmic tail of membranebound p22 phox and initiate the formation of an active and membrane-bound enzyme complex (17). PMA activates a PKC pathway independent of receptor stimulation (38) and the activated PKC phosphorylates p47 phox to form a functional NADPH oxidase (44).…”

Section: Discussionmentioning

confidence: 99%

“…A two-step RT-PCR was performed. The first strand cDNA was synthesized by adding the following components into a nuclease-free microcentrifuge tube in a final 20-l reaction volume: 1 l of oligo(dT) [12][13][14][15][16][17][18] (500 g/ml) (Invitrogen), 2 g of total RNA, 1 l of 10 mM dATP, dGTP, dCTP, and dTTP (dNTP ) mix (Invitrogen), 4 l of 5 ϫ first strand buffer, 2 l of 0.1 M DTT, 1 l of RNase inhibitor, and 1 l of Superscript II RNase H Ϫ reverse transcriptase (Invitrogen). Samples were mixed, incubated at 42°C for 60 min, and inactivated by heating at 70°C for 15 min.…”

Section: Methodsmentioning

confidence: 99%

Superoxide anion is elevated in sympathetic neurons in DOCA-salt hypertension via activation of NADPH oxidase

Dai

Chen²,

Kreulen³

2006

American Journal of Physiology-Heart and Circulatory Physiology

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Ϫ ⅐) production is elevated in sympathetic ganglion neurons and in the vasculature of hypertensive animals; however, it is not known what enzymatic pathway(s) are responsible for O 2 Ϫ ⅐ production. To determine the pathway(s) of O 2 Ϫ ⅐ production in sympathetic neurons, we examined the presence of mRNA of NADPH oxidase subunits in sympathetic ganglionic neurons and differentiated PC-12 cells. The mRNAs for NADPH oxidase subunits p47 phox , p22 phox , gp91 phox , and NOX1 were present in sympathetic neurons and PC-12 cells, whereas the NOX4 homologue was present in sympathetic neurons but not PC-12 cells. Freshly dissociated celiac ganglion neurons from normal rats and PC-12 cells produced O 2 Ϫ ⅐ when treated with the PKC activator PMA; O 2 Ϫ ⅐ production increased by 317% and 254%, respectively. The PMA-evoked increases were reduced by pretreatment with the NADPH oxidase inhibitor apocynin. These findings indicate that NADPH oxidase is the primary source of O 2 Ϫ ⅐ in sympathetic ganglion neurons. When celiac ganglia from hypertensive rats were incubated with apocynin, O 2 Ϫ ⅐ levels were reduced to the same levels as normotensive animals, indicating that NADPH oxidase activity accounted for the elevated O 2 Ϫ ⅐ levels in hypertensive animals. To test this latter finding, we compared NADPH oxidase activity in extracts of prevertebral sympathetic ganglia of DOCA-salt hypertensive rats and shamoperated rats. NADPH oxidase activities were 49.9% and 78.6% higher in sympathetic ganglia of DOCA rats compared with normotensive controls when using ␤-NADH and ␤-NADPH as substrates, respectively. Thus elevated O 2 Ϫ ⅐ levels in hypertension may be a result of the increased activity of NADPH oxidase in postganglionic sympathetic neurons. sympathetic ganglia; phorbol ester; reduced nicotinamide adenine dinucleotide phosphate oxidase; hypertension; deoxycorticosterone acetate VASCULAR ENDOTHELIAL CELLS, smooth muscle cells, and fibroblasts generate reactive oxygen species (ROS), such as O 2 Ϫ ⅐ and H 2 O 2 . ROS play critical roles in both normal and pathophysiological states of vascular cells, including the modulation of redox-sensitive signaling pathways and gene expression, or in the pathophysiology of hypertension and atherosclerosis (16,22,26,36). Several models of hypertension are associated with an increase in vascular O 2 Ϫ ⅐ (41, 42), and this increased O 2 Ϫ ⅐ may quench the endogenous vasodilator nitric oxide (NO) to cause a loss of NO bioactivity in the vessel wall and impair the endothelium-dependent vasorelaxation, resulting in hypertension (26). ROS-mediated endothelial dysfunction in ANG II-infused rats (24) and in DOCA-salt hypertensive rats (42) can be reversed by antioxidant enzyme, endothelial NO synthase (34), or superoxide dismutase (26). ROS can also induce the expression of cardiovascular-related genes, such as those for adhesion molecules and vasoactive substances. For example, the cytokine interleukin 1 activates VCAM-1 gene expression through a mechanism that is repressed ϳ90% by the antioxidant...

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“…The complex is composed of five subunits, of which two, Cyba (alias gp91phox) and Cybb (alias alias p22 phox), resides in the membrane where they form a heterodimeric flavohemoprotein-cytochrome b558. The other three subunits-Ncf1, Ncf2 (alias p67phox) and Ncf4 (alias p40phox)-are in the resting state forming a complex in the cytosol, but when activated by any of a wide variety of stimuli, the Ncf1 protein gets heavily phosphorylated and the complex migrates to the membrane to form the activated complex [22]. The Ncf1 protein is the subunit responsible for transporting the cytosolic complex to the endosomal membrane or the cell membrane during activation [23].…”

Section: The Use Of Animal Models In a Disease-to-gene Approachmentioning

confidence: 99%

Genetics of Autoimmune Diseases: A Multistep Process

Johannesson

Hultqvist

Holmdahl

Current Concepts in Autoimmunity and Chronic Inflammation

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It has so far been difficult to identify genes behind polygenic autoimmune diseases such as rheumatoid arthritis (RA), multiple sclerosis (MS), and type I diabetes (T1D). With proper animal models, some of the complexity behind these diseases can be reduced.The use of linkage analysis and positional cloning of genes in animal models for RA resulted in the identification of one of the genes regulating severity of arthritis in rats and mice, the Ncf1 gene. The Ncf1 gene encodes for the Ncf1 protein that is involved in production of free oxygen radicals through the NADPH oxidase complex, which opens up a new pathway for therapeutic treatment of inflammatory diseases. In most cases, however, a QTL is the sum effect of several genes within and outside the QTL, which make positional cloning difficult. Here we will discuss the possibilities and difficulties of gene identification in animal models of autoimmune disorders.

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Molecular Basis of Phosphorylation-Induced Activation of the NADPH Oxidase

Cited by 357 publications

References 50 publications

A Regulated Adaptor Function of p40^phox: Distinct p67^phoxMembrane Targeting by p40^phoxand by p47^phox

A Regulated Adaptor Function of p40^phox: Distinct p67^phoxMembrane Targeting by p40^phoxand by p47^phox

Superoxide anion is elevated in sympathetic neurons in DOCA-salt hypertension via activation of NADPH oxidase

Genetics of Autoimmune Diseases: A Multistep Process

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Molecular Basis of Phosphorylation-Induced Activation of the NADPH Oxidase

Cited by 357 publications

References 50 publications

A Regulated Adaptor Function of p40phox: Distinct p67phoxMembrane Targeting by p40phoxand by p47phox

A Regulated Adaptor Function of p40phox: Distinct p67phoxMembrane Targeting by p40phoxand by p47phox

Superoxide anion is elevated in sympathetic neurons in DOCA-salt hypertension via activation of NADPH oxidase

Genetics of Autoimmune Diseases: A Multistep Process

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Product

Resources

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A Regulated Adaptor Function of p40^phox: Distinct p67^phoxMembrane Targeting by p40^phoxand by p47^phox

A Regulated Adaptor Function of p40^phox: Distinct p67^phoxMembrane Targeting by p40^phoxand by p47^phox